Penicillium griseofulvum possesses two endo-(1,4)-beta-xylanase genes, PgXynA and PgXynB, belonging to family 11 glycoside hydrolases. The enzymes share 69% identity, a similar hydrolysis profile i.e. the predominant production of xylobiose and xylotriose as end products from wheat arabinoxylan and a specificity region of six potential xylose subsites, but differ in terms of catalytic efficiency which can be explained by subtle structural differences in the positioning of xylohexaose in the PgXynB model. Site-directed mutagenesis of the "thumb" region revealed structural basis of PgXynB substrate and inhibitor specificities. We produced variants displaying increased catalytic efficiency towards wheat arabinoxylan and xylo-oligosaccharides and identified specific determinants in PgXynB "thumb" region responsible for resistance to the wheat xylanase inhibitor XIP-I. Based on kinetic analysis and homology modeling, we suggested that Pro130(PgXynB), Lys131(PgXynB) and Lys132(PgXynB) hamper flexibility of the loop forming the "thumb" and interfere by steric hindrance with the inhibitor.